基础研究 Open Access
Copyright ©The Author(s) 2005. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2005-10-28; 13(20): 2413-2419
在线出版日期: 2005-10-28. doi: 10.11569/wcjd.v13.i20.2413
人CD40 ligand基因真核表达质粒的构建及对人肝癌细胞HepG2的促凋亡作用
徐智, 沈苏南, 钱晓萍, 禹立霞, 刘宝瑞
徐智, 沈苏南, 钱晓萍, 禹立霞, 刘宝瑞, 南京大学医学院附属鼓楼医院肿瘤中心 江苏省南京市 210008
徐智, 女, 1980-06-19生, 江苏省南京市人, 汉族, 2003年南京大学医学院肿瘤学硕士生.
基金项目: 国家自然科学基金资助项目, No. 30471701.
通讯作者: 刘宝瑞, 210008, 江苏省南京市中山路321号, 南京大学医学院附属鼓楼医院肿瘤中心. baoruiliu@medmail.com.cn
电话: 025-83106666-76382
收稿日期: 2005-08-29
修回日期: 2005-09-01
接受日期: 2005-09-06
在线出版日期: 2005-10-28

目的: 构建含人CD40 ligand(CD40L)基因的真核表达载体, 并使之在人肝癌细胞HepG2中表达, 研究CD40L稳定表达对HepG2细胞的影响.

方法: 从人外周血单个核细胞总RNA中经RT-PCR扩增出人CD40L基因, 经过酶切连接进入真核表达载体pcDNATM3.1/myc-His(-)A制备重组质粒. 重组质粒经酶切及测序证实人CD40L基因成功克隆进入真核表达载体pcDNATM3.1/myc-His(-)A, 并进一步转入大肠杆菌(E.coli DH5α)大量扩增. 实验分4组, A组HepG2细胞转染重组质粒, B组HepG2细胞转染不含CD40L cDNA的空质粒, C组HepG2细胞正常培养, D组HepG2细胞加入G418作为转染对照. RT-PCR及流式细胞术鉴定A, B, C 3组细胞表面CD40L和CD40的表达后, A, B, C 3组细胞分别设6个复孔培养72 h后, 流式细胞术检测细胞凋亡、细胞周期分布和Fas表达.

结果: 测序结果显示人CD40L基因真核表达质粒CD40L-pcDNATM3.1/myc-His(-)A构建成功. 流式细胞术检测A组HepG2细胞表面CD40L表达为39.7%, CD40表达为15.4%; B和C组细胞表面仅有CD40表达, 分别为31.7%和28.5%. A组细胞凋亡率45.0±0.3%, B, C组均未发生明显凋亡(P<0.01). 与C组细胞比较, A组细胞周期分布主要阻滞在G1期(90.4±1.3% vs 60.6±1.5%, P<0.01), S期(6.32±1.0% vs 12.0±0.7%)和G2/M期分布(3.3±0.7% vs 27.3±1.2%)均有减少(P<0.01). A组细胞Fas表达率(27.8±1.5%)较B组(3.2±0.8%)和C组(4.2±1.0%)明显上调(P<0.01).

结论: 我们构建的人CD40L基因真核表达质粒CD40L-pcDNATM3.1/myc-His(-)A可以在人肝癌细胞HepG2中稳定表达, CD40L对于HepG2细胞具有促凋亡的作用, 这可能与CD40L-CD40作用后导致HepG2细胞Fas表达上调和细胞周期阻滞有关.

关键词: 肝癌; CD40L; 质粒; 真核表达载体
引文著录: 徐智, 沈苏南, 钱晓萍, 禹立霞, 刘宝瑞. 人CD40 ligand基因真核表达质粒的构建及对人肝癌细胞HepG2的促凋亡作用. 世界华人消化杂志 2005; 13(20): 2413-2419
Construction and expression of a eukaryotic expression plasmid containing human CD40 ligand in human hepatocellular carcinoma cell line HepG2 and its effect on apoptosis
Zhi Xu, Su-Nan Shen, Xiao-Ping Qian, Li-Xia Yu, Bao-Rui Liu
Zhi Xu, Su-Nan Shen, Xiao-Ping Qian, Li-Xia Yu, Bao-Rui Liu, Cancer Center of Drum Tower Hospital, Medical School of Nanjing University, Nanjing 210008, Jiangsu Province, China
Supported by: National Natural Science Foundation of China, No. 30471701.
Correspondence to: Professor Bao-Rui Liu, Cancer Center of Drum Tower Hospital, Medical School of Nanjing University, 321 Zhongshan Road, Nanjing 210008, Jiangsu Province, China. baoruiliu@medmail.com.cn
Received: August 29, 2005
Revised: September 1, 2005
Accepted: September 6, 2005
Published online: October 28, 2005

AIM: To construct and express a eukaryotic expression plasmid containing human CD40 ligand (CD40L) in human hepatocellular carcinoma cell line HepG2 for the biological function study of CD40L on HepG2 cells.

METHODS: Human CD40L cDNA was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) with the specific primers from the RNA of human peripheral blood monocyte (PBMC) and directly ligated into the eukaryotic expression vector pcDNATM3.1/myc-His(-)A through digestion with specific restriction endonuclease. The recombined plasmid was transformed into the E. coli DH5α to amplify CD40L gene. Then HepG2 cells were divided into 4 groups. The cells in group A were transfected with the recombined plasmid, and group B with the blank plasmid (not containing CD40L cDNA). The cells in group C were just normally cultured, and the ones in group D weren't transfected but added G418 as the control for transfection. The expression of CD40L and CD40 were detected by RT-PCR and fluorescent activated cell sorter (FACS) in HepG2 cells, and the apoptosis, cell cycle, as well as Fas expression, were measured by FACS.

RESULTS: The full-length human CD40L cDNA was successfully cloned into the eukaryotic vector pcDNATM3.1/myc-His(-)A. The recombinant plasmid was stably transfected into group A and the rate of CD40L expression was 39.7%. There was no CD40L expression in group B and C. The rates of CD40 expression in group A, B and C were 15.4%, 31.7% and 28.5%, respectively. The apoptotic rate of group A was 45.0±0.3%, but neither group B or C showed obvious apoptosis (P <0.01). Compared with group C, a larger proportion of cells in group A was restrained at G0/G1 phase (90.4±1.3% vs 60.6±1.5%, P <0.01), while the proportions of the cells in S phase (6.32±1.0% vs 12.0±0.7%) and G2/M phase (3.3±0.7% vs 27.3±1.2%) were reduced (both P <0.01). The expression of Fas in group A showed marked increase as compared with that in group B and C (27.8±1.5% vs 3.2±0.8%, 4.2±1.0%, respectively, both P <0.01).

CONCLUSION: The eukaryotic expression plasmid pcDNATM3.1/myc-His(-)A can be stably expressed in HepG2 cell line. CD40L-induced apoptosis of HepG2 is correlated with the increased expression of Fas and cell cycle arrest.

Key Words: Hepatocellular carcinoma; CD40 ligand; Plasmid; Eukaryotic expression vector
0 引言

原发性肝癌(HCC)是最常见的恶性肿瘤, 除手术切除外对放疗和化疗多不敏感. 日本临床研究发现CD3mAb激活的淋巴细胞治疗手术后HCC患者与手术后未行免疫治疗的患者相比, 无病生存时间获得显著延长[1]. 提示免疫基因疗法(immunogenetic therapy)在肝癌的治疗中具有潜力. CD40 ligand(CD40L), 属于肿瘤坏死因子(TNF)基因超家族, 主要在活化的T淋巴细胞表面表达. 转基因表达CD40L的细胞联合其他细胞因子可刺激B细胞体外大量扩增, 使之成为丰富的抗原递呈细胞(APC), 在B细胞瘤苗制备及特异性免疫治疗领域发挥重要作用[2-5]. 我们在肝癌特异性B细胞瘤苗的制备中, 构建了含有人CD40L的真核表达载体, 并使其在人肝癌细胞HepG2中稳定表达, 发现CD40L对于HepG2细胞有促进凋亡的作用, 并对这一作用机制进行探讨.

1 材料和方法
1.1 材料

人肝癌HepG2细胞株、大肠杆菌E.coli DH5α由本实验室提供; 健康人外周血来源于南京市血液中心; 淋巴细胞分离液(Ficoll, 上海生化试剂二厂); pcDNATM3.1/myc-His(-)A、RPMI 1640培养液、Trizol试剂盒和LipofectamineTM 2000(Invitrogen公司); 小牛血清(FCS, 杭州四季青公司); M-MLV RTase cDNA Synthesis试剂盒、DL 2000 marker、BamHⅠ、EcoRⅠ、BglⅡ和HindⅢ(Takara公司); Taq酶和T4连接酶(Promega公司); 小量胶回收试剂盒(上海华舜生物公司); 鼠抗人FITC-CD40LmAb, 鼠抗人FITC-CD40mAb, 鼠抗人PE-FasmAb, Annexin V: PE Apoptosis Detection KitⅠ和CycleTEST PlusTM试剂盒(BD公司). 引物合成和测序由上海博亚生物公司完成.

1.2 方法

使用淋巴细胞分离液常规分离人外周血单个核细胞(PBMC). RPMI 1640, 100 mL/L FCS, 4 mg/L植物血凝素培养PBMC 12 h后收集细胞, 用Trizol抽提总RNA, M-MLV RTase cDNA Synthesis试剂盒制备cDNA文库. PCR扩增cDNA文库中CD40L cDNA. 引物f: CG GAA TTC GCC ACC ATG ATC GAA ACA TAC AAC C, r: CCG GGA TCC TCA GAG TTT GAG TAA GCC AAAG. 反应条件: 95℃ 5 min, 94℃ 30 s, 64℃ 45 s, 72℃ 1 min, 循环35次, 72℃ 10 min. 扩增后的CD40L cDNA和pcDNATM3.1/myc-His(-)A分别经过EcoRⅠ和BamHⅠ酶切后使用琼脂糖电泳, 小量胶回收试剂盒回收带有BamHⅠ和EcoRⅠ酶切位点的CD40L cDNA和pcDNATM3.1/myc-His(-)A, T4连接酶连接回收片断获得重组质粒. 重组质粒转化大肠杆菌E.coli DH5α, 在50 mg/L氨苄青霉素LB培养板上培养并筛选阳性克隆细菌. 挑取10个阳性克隆细菌经SDS碱裂解法抽提重组质粒后分别行EcoRⅠ和BamHⅠ, BglⅡ和EcoRⅠ以及Hind Ⅲ酶切鉴定. 3种酶切结果均与预计相符的重组质粒通过ABI 3730自动测序仪测序鉴定. 使用LipofectamineTM 2000转染HepG2细胞, 实验分4组: A组: 测序正确的重组质粒CD40L-pcDNATM3.1/myc-His(-)A转染HepG2细胞; B组: 不含CD40L的空质粒pcDNATM3.1/myc-His(-)A转染HepG2细胞; C组: HepG2细胞单纯培养; D组: HepG2细胞加入G418. 待细胞生长融合达90%后转染, 按4 mg质粒和10 mL LipofectamineTM 2000的比例制备脂质体DNA复合物转染细胞, 转染后24 h更换含100 mL/L FCS的RPMI 1640完全培养液, 48 h后1∶10稀释A, B, D组细胞并加入300 mg/L G418加压筛选, C组细胞继续单纯培养. 10 d后, D组细胞完全死亡, A, B组细胞形成具有G418抗性基因的克隆, 继续在96孔板中培养单克隆细胞并扩增. 使用Trizol抽提A, B, C组细胞中总RNA, 经RT-PCR扩增后琼脂糖电泳PCR产物. 引物使用f和r, 反应条件同上. A, B, C组细胞各取1×105个, 分别混合鼠抗人FITC-CD40LmAb和鼠抗人FITC-CD40mAb各10 mL, 室温避光孵育30 min, PBS洗涤2遍, 流式细胞仪(FACS)检测CD40L和CD40表达. 3组细胞各以2×105个/孔接种在6孔板内, 每组设6个复孔, 100 mL/L FCS RPMI 1640培养液在50 mL/L CO2, 37℃培养箱中培养72 h, 48 h后换液. 培养72 h后的3组细胞各复孔分别使用Annexin V: PE Apoptosis Detection KitⅠ, CycleTEST PlusTM试剂盒和鼠抗人E-FasmAb经流式细胞仪检测凋亡, 细胞周期分布和Fas表达, 实验按说明书操作.

统计学处理 使用方差分析和t检验.

2 结果

CD40L cDNA接入在pcDNATM3.1/myc-His(-)A多克隆位点EcoRⅠ和BamHⅠ之间, EcoRⅠ和BamHⅠ酶切结果显示pcDNATM3.1/myc-His(-)A长约5.5 kb, CD40L cDNA长约800 bp; BglⅡ和EcoRⅠ为CD40L-pcDNATM3.1/myc-His(-)A内在唯一限制性内切酶位点, BglⅡ和EcoRⅠ之间约为1.7 kb, 其余约为4.5 kb; Hind Ⅲ为CD40L cDNA和pcDNATM3.1/myc-His(-)A内部各含有的一个限制性内切酶位点, 之间长度约为300 bp, 其余约为6 kb(图1). 测序结果显示CD40L cDNA序列与NIH公布的CD40L cDNA序列一致, 并且接入方向正确(图2). RT-PCR后电泳结果显示所得PCR产物长度约为800 bp, 与CD40L cDNA长度一致, 而转染空质粒和未转染的HepG2细胞总RNA经RT-PCR后没有CD40L cDNA(图3). A组HepG2细胞表面有CD40L和CD40稳定表达, 其中CD40L表达为39.7%, CD40表达为15.4%; B, C组细胞表面仅有CD40表达, 分别为31.7%和28.5%(图4). A组细胞凋亡率45.0±0.3%, B和C组均未发生明显凋亡(P<0.01)(图5和表1). A组细胞周期G0/G1, S和G2/M分别为90.4±1.3%, 6.32±1.0%, 3.3±0.7%, C组分别为60.6±1.5%, 12.0±0.7%, 27.3±1.2%(图6). A组与C组相比, 细胞周期主要阻滞在G0/G1期(P<0.01), S和G2/M期分布减少(P<0.01)(表2). A, B, C组细胞Fas表达率分别为27.8±1.5%, 3.2±0.8%和4.2±1.0%(图7), A组表达率明显高于B, C组(P<0.01)(表3).

表1 3组细胞凋亡率(%, mean±SD).
ABC
凋亡率45.0±0.3b0.1±0.10.1±0.0
bP<0.01 vs B组和C组.
图1
图1 酶切检测各克隆质粒结果. A: EcoRⅠ和BamHⅠ酶切结果; B: Bgl Ⅱ和EcoRⅠ酶切结果; C: Hind Ⅲ酶切结果; M: DL 2000 marker (下同).
表2 细胞周期分布(%, mean±SD).
AC
G0/G190.4±1.3b60.6±1.5
S6.32±1.0d12.0±0.7
G2/M3.3±0.7f27.3±1.2
bP<0.01,
dP<0.01,
fP<0.01 vs C组.
图2
图2 测序检测重组质粒结果.
表3 3组细胞Fas表达率(%, mean±SD).
ABC
Fas表达率27.8±1.5b3.2±0.84.2±1.0
bP<0.01 vs B组和C组.
图3
图3 RT-PCR检测3组细胞CD40L mRNA表达结果. 1: 重组质粒CD40L-pcDNATM3.1/myc-His(-)A转染的HepG2细胞; 2: 空质粒pcDNATM3.1/myc-His(-)A转染HepG2细胞; 3: HepG2细胞(下同).
图4
图4 FACS检测3组细胞CD40和CD40L结果.
图5
图5 FACS检测3组细胞凋亡结果.
图6
图6 FACS检测A, C组细胞周期分布. A: A组; B: C组.
图7
图7 FACS检测3组细胞Fas结果.
3 讨论

CD40L是35 ku的Ⅱ型膜蛋白, 属于肿瘤坏死因子(TNF)家族, 可以短暂表达在成熟的CD4+T细胞、CD8+T细胞亚群、γδT细胞、肥大细胞、IL-2激活的NK细胞等多种细胞表面. CD40L与CD40胞外区结合后, CD40形成多聚体复合物激活NF-κB(nuclear factor kappa of B cells)或经过TRAF家族分子、JAK3、STAT3等信号途径活化第二信使, 进一步调控核内基因转录[6]. 在正常B细胞中, CD40L-CD40通过NF-κB上调IL-6、黏附分子(CD54)和抗凋亡蛋白(cIAP1, cIAP2, Bcl-xL, A1等)表达[7,8], 促使B细胞增殖活化. 在T细胞中, CD40L-CD40上调TRAF1表达进而抑制CD8+T细胞凋亡[9]. 目前CD40L-CD40对肿瘤细胞生长调控机制尚未明确: 对于Hodgkin淋巴瘤, 低分化B细胞恶性肿瘤(如滤泡细胞淋巴瘤)等, CD40L-CD40可以导致肿瘤增殖、耐受化疗; 对于HIV相关淋巴瘤, CD40L-CD40活化的NF-κB促进病毒复制, 加速肿瘤的生长; 对于非HIV淋巴瘤, CD40L-CD40导致TRAF的活化促使其他TNFR家族成员(如Fas等)表达, 诱导细胞进入凋亡相关级联反应途径, 抑制肿瘤细胞生长[10-13].

我们发现, 转染重组质粒的HepG2细胞与对照组细胞(转染空质粒和未转染的HepG2细胞)相比CD40的表达发生下调, 推测这一变化可能与CD40L-CD40相互作用有关, 或由于CD40L基因在转录过程中产生了影响CD40转录的信号分子. CD40L在HepG2细胞中表达后对CD40表达影响的具体调控机制尚有待进一步研究. 我们构建的CD40L-pcDNATM3.1/myc-His(-)A可以在人肝癌细胞HepG2中稳定表达, 转染重组质粒的HepG2在表达CD40和CD40L的同时出现凋亡现象, 而对照组细胞在相同培养条件下均未出现凋亡, 说明pcDNATM3.1/myc-His(-)A对于HepG2细胞本身并不具有促凋亡作用, 而是由于CD40L-CD40相互作用而导致凋亡发生. 目前研究显示CD40L可以通过细胞周期阻断和/或诱导凋亡对多种肿瘤细胞(如乳腺癌、非小细胞肺癌、宫颈癌、膀胱癌、胶质细胞瘤等)产生生长抑制效应, 但是对于其相应的正常细胞并没有明显的副作用[14-21]. 本研究结果表明稳定表达CD40L的HepG2细胞, Fas表达上调, 细胞周期主要阻滞在G0/G1期, S期和G2/M期分布减少, 推断CD40L对HepG2的生长调控机制可能是通过CD40L-CD40作用促使TNFR家族成员Fas表达并阻断细胞生长周期, 诱导凋亡的发生.

目前已有研究证实将稳定表达CD40L的NIH3T3细胞作为刺激源活化DC与既往使用GM-CSF和IL-4培养方法相比, CD40L活化的DC MHCⅡ类分子、CD80和IL-12的表达更高, 可以在体内产生更强的激活CTL能力[22-27]. 另有研究报道CD40L可以持续扩增B细胞达56 d之久, 使之高表达MHCⅠ, MHCⅡ, CD80, CD86等抗原递呈必需的分子, 并有效提呈肿瘤细胞裂解物、抗原肽、抗原cDNA甚至RNA, 诱发特异性细胞免疫反应[28-30]. 我们构建的稳定表达CD40L的HepG2细胞是否具有同样活化DC或B细胞的能力, 同时CD40L作为细胞免疫应答中关键的共刺激分子, 能否增强HepG2细胞作为抗原的免疫原性, 这些表达CD40L的HepG2细胞和凋亡细胞可否成为更有利于APC提呈的抗原, 将是我们深入研究的方向. 总之, 人CD40L基因真核表达质粒CD40L-pcDNATM3.1/myc-His(-)A可以在人肝癌细胞HepG2中稳定表达, CD40L的稳定表达引起HepG2细胞CD40表达下调、Fas表达上调和细胞周期阻滞在G1期, 产生促凋亡作用. 以上结果将有助于今后对CD40L在肝癌免疫治疗中的应用进行深入研究.

电编: 张敏 编辑: 潘伯荣 审读: 张海宁

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